As the dopamine transporter (DAT) is the primary mechanistic target for cocaine's actions, the continued development of molecular tools with which to study this protein, both in vivo and at the molecular level, is our primary objective. In this project we aim to discover and develop potential medications for treatment of cocaine abuse and other related neuropsychiatric disorders, as well as understanding how drug-receptor interactions at the DAT contribute to both cocaine's actions and the actions of our own ligands, that do not produce a cocaine-like behavioral profile in animal models. In a continued effort to develop structure-activity relationships (SAR) for the benztropine-based DAT inhibitors and to optimize these ligands as in vivo tools, we have synthesized and evaluated novel analogues using this design strategy. All of these compounds have been evaluated in vivo for binding and function at the DAT as well as selectivity over the serotonin and norepinephrine transporters and other CNS receptor systems that may contribute to their behavioral profile. The compounds that show high affinity and selectivity for the DAT have then been further evaluated in vivo, using numerous animal models of cocaine abuse. Thus far the combination of these studies has led us to hypothesize that although the benztropines bind to the DAT and elevate dopamine levels to concentrations that can exceed those produced by cocaine, because the increase is considerably slower than cocaine, compensatory mechanisms may be contributing to the lack of cocaine-like effects for these drugs. We are currently continuing to tweak our molecules to optimize their pharmacological actions in vivo and follow their development toward the clinic. Concurrently, we are using these compounds and their derivatives as molecular tools, in combination with site-directed mutagenesis and computational analysis to form hypotheses on how cocaine and the benztropines bind to the DAT and how that may be similar or different from one another and from the substrate dopamine. The recent crystal structure of the bacterial homologue LeuT has provided an important template for molecular modeling studies and our experimental evidence suggests that these compounds bind the DAT at overlapping sites but that unique conformational changes induced by cocaine may be relevant to its pharmacological profile, in vivo.

Summary:

The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus or squirrel monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) is directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics and may be directly related to their lack of cocaine-like behavioral profiles. Further investigation into correlating structure, pharmacological action and pharmacodynamics of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have further characterized one of our novel DAT ligands, JHW 007 (N-n-butyl-44-diF-BZT), and discovered that this compound has a unique pharmacological profile in that it slowly associates with the dopamine transporter and not only demonstrates no cocaine-like actions in mice, rats or monkeys, but fully blocks cocaine-induced locomotor activity and the cocaine discriminative stimulus in mice. These studies are the first to show that a dopamine uptake inhibitor can prevent cocaine from producing its psychostimulant actions and further support the hypothesis that the rate of in vivo occupancy of the DAT can dictate the behavioral actions of these compounds. Further evaluation of this and other N-substituted BZTs using microdialysis has allowed us to relate the rate and levels of increasing extracellular dopamine, in vivo, with binding of these compounds to the DAT. Further investigation has shown that several additional analogues of JHW007 show similar profiles in vivo and have been identified as lead candidates for development as medications to treat cocaine abuse, as well as ADHD. Recent studies using site-directed mutagenesis have revealed differences in binding domains between the BZTs, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, experimental evidence using the DAT inhibitors cocaine, WIN 35,428, and several benztropine analogues and comparing them to the substrates dopamine and MDMA has provided evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Radioiodinated analogues of both azido (photoactivated) and isothiocyanato-derivatives of our tropane based DAT inhibitors have been synthesized and are currently being elucidate transmembrane domains at which these compounds bind covalently to both DAT and SERT. We have also synthesized several high affinity fluorescent DAT ligand that are currently being used to characterize the trafficking of DAT in living neuronal cells and as a fluoroprobe for visualization of dopamine neurons in stem cells.